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and Differentiation (2009) 16, 321–330 & 2009 Macmillan Publishers Limited All rights reserved 1350-9047/09 $32.00 www.nature.com/cdd The Aurora B activity is required for the maintenance of the differentiated state of murine myoblasts

G Amabile1,2, AM D’Alise1, M Iovino1, P Jones3, S Santaguida4, A Musacchio4, S Taylor5 and R Cortese*,1

Reversine is a synthetic molecule capable of inducing dedifferentiation of C2C12, a murine myoblast cell line, into multipotent progenitor cells, which can be redirected to differentiate in nonmuscle cell types under appropriate conditions. Reversine is also a potent inhibitor of Aurora B, a kinase required for mitotic segregation, spindle checkpoint function, and H3 phosphorylation, raising the possibility that the dedifferentiation capability of reversine is mediated through the inhibition of Aurora B. Indeed, here we show that several other well-characterized Aurora B inhibitors are capable of dedifferentiating C2C12 myoblasts. Significantly, expressing drug-resistant Aurora B mutants, which are insensitive to reversine block the dedifferentiation process, indicating that activity is required to maintain the differentiated state. We show that the inhibition of the spindle checkpoint or cytokinesis per se is not sufficient for dedifferentiation. Rather, our data support a model whereby changes in histone H3 phosphorylation result in remodeling, which in turn restores the multipotent state. Cell Death and Differentiation (2009) 16, 321–330; doi:10.1038/cdd.2008.156; published online 31 October 2008

Lineage-restricted cells can be reprogramed to a state Evidence is emerging that the role of Aurora B is not of pluripotency by several different manipulations, including restricted to and cell division. A key substrate of somatic cell nuclear transfer1 embryonic stem cell (ES) Aurora B is 10 (ser10) of histone H3, and although this fusion 2 or even by cotransfection of four specific transcription phosphorylation event was originally thought to be involved in factors. 3–5 In particular, C2C12 myoblasts can be repro- chromosome condensation, it now appears that it dissociates gramed to dedifferentiate into multipotent progenitor cells HP1 from methylated histone H3 (K9me3) at the that can be redirected to other cell types under appropriate onset of mitosis.13,14 Indeed, histone H3 (ser10) is an stimuli, including the ectopic expression of Msx1 gene,6 important element in the combinatorial histone code that is or by exposure to chemical compounds such as associated with both active and inactive chromatin.15 At the reversine.7 onset of mitosis, phospho-Ser10-H3 is responsible for the Reversine, a purine derivative, was identified by virtue of its ejection of HP1 proteins from their on chromatin, ability to increase the plasticity of C2C12 myoblasts. This probably through a steric hindrance.13,14 Furthermore, it was compound interferes with the normal differentiation pathway demonstrated that the modifications of histone H3 and causing C2C12 cells to dedifferentiate to a pluripotent state. consequently the redistribution of HP1s are linked to More recently, it was demonstrated that reversine can chromatin reorganization and to cellular dedifferentation.16 differentiate human fibroblasts into skeletal muscle cells in Consistent with a role for Aurora B in chromatin structure, it vitro and in vivo,8 suggesting that reversine is capable of also was recently shown that Aurora B is required to remodel reprograming primary somatic cells to a state of increased chromatin during postmitotic cell differentiation of mesen- plasticity. We recently discovered that reversine is also a chymal stem cells and in the transition of to plasma cells.17 potent inhibitor.9 The Auroras are serine/ The ability of reversine to both dedifferentiate C2C12 cells threonine required for multiple aspects of mitosis in and to inhibit Aurora B raised the possibility that its eukaryotic cells. Aurora A promotes maturation dedifferentiation capability is mediated through Aurora B. and spindle assembly,10 whereas Aurora B is required for Here, we present the evidence that in addition to reversine, histone H3 phosphorylation, chromosome segregation,11 the several other Aurora B inhibitors can dedifferentiate C2C12 spindle assembly checkpoint and cytokinesis.12 myoblasts. By using drug-resistant Aurora B mutants, we

1CEINGE Biotecnologie Avanzate, Naples, Italy; 2Department of Cellular Biotechnology and Hematology, University of Rome La Sapienza, Rome, Italy; 3IRBM Merck Research Laboratory, Pomezia, Italy; 4Department of Experimental Oncology, European Institute of Oncology, Milan, Italy and 5Faculty of Life Sciences, University of Manchester, Manchester, UK *Corresponding author: R Cortese, Medical Biotechnology, CEINGE Biotecnologie Avanzate, Via Comunale Margherita 482, Naples 80131, Italy. Tel: þ 39 335 81 04 832; Fax: þ 39 081 746 36 50; E-mail: [email protected] Keywords: dedifferentiation; reversine; Aurora B; myoblasts Abbreviations: ES, embryonic stem cells; ap2, adipocyte fatty acid-binding protein; LPL, lipoprotein lipase ; ALP, alkaline phosphatase; NMMII, nonmuscle II heavy chain ; MEK, mitogen-activated extracellular signal-regulated kinase; ADM, adipogenic-differentiating medium; ODM, osteogenic-differentiating medium; MRFs, muscle regulatory factors; Id(s), inhibitor of differentiation(s); Myf5, myogenic factor 5; Lys, lysine; Ser, serine; p, phospho; me, methyl Received 18.6.08; revised 19.9.08; accepted 19.9.08; Edited by G Cossu; published online 31.10.08 Aurora B inhibition induces dedifferentiation G Amabile et al 322

provide compelling evidence that the effects of reversine are should have different spectra of off-target effects.19 We mediated through Aurora B as opposed to an off-target effect. chose to use , which is relatively selective for Furthermore, we demonstrate that reversine remodels the Aurora B,20 VX-680, which is a dual Aurora A/B inhibitor21 chromatin associated with several genes that are induced or and MLN-8054, which is relatively selective for Aurora A.22 repressed during the phenomenon of dedifferentiation. Significantly, hesperadin, VX-680 and MLN-8054 induced Altogether, our data suggest a novel role for the Aurora B dedifferentiation of C2C12 myoblasts (Figure 1). C2C12 cells kinase in gene regulation and in the maintenance the were treated with compounds for 72 h as indicated; after this, differentiated state. the drug-containing media were removed and the cells were cultured in drug-free medium suitable for the development of adipocytes (adipogenic-differentiating medium; ADM) and Results and Discussion osteoblasts (osteogenic-differentiating medium; ODM), respectively. After 7 days, cells were stained with Oil Red Aurora kinase inhibitors reprogram C2C12 myo- O reagent to evaluate the presence of adipocytes or for blasts. Reversine inhibits several proteins including MEK1, alkaline phosphatase to evaluate the presence of osteoblasts nonmuscle myosin II (NMMII), Aurora A and B.9 On the basis (Figure 1a). In addition, to have a quantitative analysis of the of several indirect lines of evidence, it was suggested that dedifferentiation phenomenon, we used a fluorescent reversine induces dedifferentiation by the simultaneous substrate system to evaluate the amount of lipid droplets in inhibition of MEK1 and of NMMII.18 However, as reversine the case of adipocytes and the alkaline phosphatase activity is a potent , we wanted to determine whether in the case of osteoblasts (Figure 1b, see Materials and the inhibition of Aurora A and/or B may be involved in the Methods also). dedifferentiation process. To do this, we asked whether a Furthermore, we decided to investigate also the expression panel of other Aurora kinase inhibitors could dedifferentiate of specific markers of both osteoblast and adipocyte C2C12 cells, reasoning that structurally diverse inhibitors differentiation. In particular, after the treatment with 500 nM

ControlReversine VX-680 Hesperadin Oil Red staining ALP staining

72 hr treatment (500 nm)

ALP activity Adipo Red assay 4500 4500 4000 10000 4000 12000 3500 3500 10000 3000 8000 3000 2500 8000 2500 6000 RFU 2000 2000 6000 1500 RFU 4000 1500 4000 1000 1000 2000 2000 500 500 0 0 0 0

Control VX-680 Control VX-680 Control VX-680 Control VX-680 Reversine Reversine Reversine Reversine Hesperadin MLN 8054 Hesperadin MLN 8054 Hesperadin MLN 8054 Hesperadin MLN 8054 72 hr treatment (500 nm) 72 hr treatment (50 nm) 72 hr treatment (500 nm) 72 hr treatment (50 nm) Figure 1 Aurora kinases inhibitors induce C2C12 dedifferentiation. (a) C2C12 cells were treated for 72 h with 500 nM of reversine, hesperadin and VX-680. Alkaline phoshatase and Oil Red O stainings were performed after 7 days of exposure to osteogenic-differentiating (ODM) and to adipogenic-differentiating media (ADM), respectively. The arrows indicate cells positive for Alkaline phosphatase (upper panel) and for lipid droplets content (lower panel) (b) C2C12 cells were treated for 72 h with 500 or 50 nM of reversine, hesperadin, VX-680 and MLN-8054. Then, cells were cultured in ODM and ADM media as indicated. ALP activity of C2C12 cells after the treatment was assayed using a fluorescent substrate system and the results were expressed in relative fluorescence units (RFUs). Quantitative analysis of adipocyte differentation was assayed with AdipoRed reagent to evaluate the lipids droplets content. The histograms show the RFUs measured with excitation at 485 nm and an emission at 572 nm

Cell Death and Differentiation Aurora B inhibition induces dedifferentiation G Amabile et al 323 of each compound and the exposure for 1 week to observations, and Manfredi et al.22 and Girdler et al.23 Thus, differentiating media, we analyzed the expression of osteo- the spectrum of inhibitor potencies suggests that Aurora B pontin and collagen type 1 for the osteoblasts and the kinase rather than A might be the target of reversine in the expression of the adipocyted fatty acid-binding protein (ap2) dedifferentiation program. and of lipoprotein lipase (LPL) for the adipocytes. In Figure 2, Importantly, although reversine inhibits MEK1 and we show that the treatment with the Aurora kinase inhibitors NMMII,18 hesperadin and VX680 are not particularly potent caused a relevant increase of these markers strengthening against these two (Supplementary Figure 1), the notion that reversine-treated myoblasts can be induced to indicating that the inhibition of MEK1 and NMMII is not differentiate into other cell types. necessary for C2C12 dedifferentiation. Among the Aurora kinase inhibitors tested, reversine and hesperadin were very potent inducers of dedifferentiation, Drug-resistant Aurora B kinase mutants block the whereas MLN-8054 and VX-680 were only active at sig- dedifferentiation capabilities of reversine . The fact that nificantly higher concentration (Figure 1b). Interestingly, multiple inhibitors of Aurora B can dedifferentiate C2C12 reversine and hesperadin are low nanomolar inhibitors of cells is consistent with the notion that Aurora B activity is Aurora B kinase (S Santaguida and A Musacchio, unpub- required to maintain the myoblasts in the differentiated state. lished observations, and Figure 3), and their effect on cultured However, although unlikely, we cannot rule out the possibility cells is mainly because of the inhibition of Aurora B kinase. By that all these inhibitors share a common ‘off-target’ . contrast, when using VX-680 and MLN-8054, on cultured Therefore, to exclude this possibility, we set out to express cells, the predominant phenotype from these drugs is the drug-resistant Aurora B mutants in C2C12 cells and ask appearance of monopolar spindles, a hallmark of Aurora A whether this expression blocked reversine-induced inhibition (A Musacchio and S Santaguida, unpublished dedifferentiation. We reasoned that if the dedifferentiation

Osteopontin Collagen Type 1 24 40

20 30 16

12 20

Fold change 8 Fold change 10 4

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Control VX-680 Control VX-680 Reversine Reversine Hesperadin Hesperadin

Adipocyte fatty acid-binding protein (ap2) Lipoprotein lipase (LpL)

25 12

20 10 8 15 6 10 Fold change

Fold change 4 5 2

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Control Control VX-680 VX-680 Reversine Reversine Hesperadin Hesperadin Figure 2 The use of Aurora kinases inhibitor induces the initiation of osteogenic and adipogenic program. (a) qRT-PCR for osteogenic (osteopontin and collagen type 1) and (b) adipogenic (adipocyte fatty acid-binding protein and lipoprotein lipase) genes was performed on C2C12 cells treated with 500 nM reversine, VX-680 and hesperadine, and then cultured in osteogenic-differentiating (ODM) and adipogenic-differentiating media (ADM) (as described in Materials and Method section). Genes mRNA levels were normalized using GAPDH. Untreated C2C12 cells were used to calculate the fold enrichment

Cell Death and Differentiation Aurora B inhibition induces dedifferentiation G Amabile et al 324

nM M

0 1 5 10 50 100 500 1 5 10 50 100 500 Reversine [32P]-H3 h-Aurora B WT Coomassie

[32P]-H3 h-Aurora B G160V Coomassie

Control AuroraB WTAurora B Y156HAurora B G160V 42 kDa anti-Myc-Tag 36 kDa

h-Aurora B

m-Aurora B

Actin

C2C12 cells Figure 3 The Aurora B G160V mutant is resistant to reversine. Human Aurora B:INCENP IN-box wild-type (WT; upper panels) and G160V mutant (lower panel) were incubated at 301C for 1 h with histone H3 as a substrate, the indicated growing concentrations of reversine, and [g-32P]-ATP. DMSO was used as control. The incorporation of radioactive phosphate on histone H3 was visualized by autoradiography. (a) C2C12 cells were transfected with Aurora B WT, Aurora B Y156H and Aurora B G160V. The presence of ectopic was assessed after 48 h post-trasfection by western blot analysis using both an anti-Myc-tag and anti-Aurora B antibody. An antiactin antibody was used as loading control. (b) C2C12 cells were transfected with Aurora B WT, Aurora B Y156H and Aurora B G160V as indicated. After 48 h, cells were treated with reversine 50 nM for further 72 h and typical western blot was performed to evaluate the protein levels of ectopic genes

capability of reversine was mediated through the inhibition of western blot analysis showed expression of the exogenous Aurora B, the ectopic expression of a drug-resistant Aurora B Aurora B proteins (Figure 3b). In a parallel experiment, 48-h mutant should maintain the myoblast state despite the post-transfection, 50 nM reversine was added for 72 h. We presence of reversine. By contrast, if reversine effects were observed that reversine efficiently dedifferentiated the non- through another target, the expression of the drug-resistant transfected controls as shown by the change of cell Aurora B mutants should have no effect. morphology (Figure 4a). Significantly, C2C12 cells trans- Recently, we have described two mutations in the active fected with the drug-resistant mutants did not acquire the site of Aurora B, namely G160V and Y156H, which render it round morphology typical of the controls (Figure 4a). In resistant to ZM447439, hesperadin, VX-680 and MLN-8054.23 particular, a myotube formation assay was performed to better On the basis of the 3D structure of the complex between investigate whether the mutant G160V was sufficient to inhibit reversine and Aurora B,9 we reasoned that these mutants the dedifferentiation capabilities of reversine. After 72 h of should also be resistant to reversine. To prove this prediction, reversine treatment, the cells were kept in the same plate for 8 we tested the ability of increasing concentrations of reversine days replacing the medium each 48 h to permit the myotubes to inhibit the activity of recombinant wild-type (WT) human formation. We observed that only the control cells and the Aurora B:INCENP IN-box complex, or of equivalent com- cells previously transfected with the resistant mutant were plexes bearing the G160V (Figure 3a) or the Y156H mutation able to form myotubes after reversine exposure. We found (data not shown), using histone H3 as an in vitro substrate. In that after the transfection with the mutant, about 70% of cells agreement with our prediction, the G160V and Y156H gave rise myotubes. Not trasfected cells and cells trasfected mutations conferred a B100-fold or higher resistance to with Aurora B WT lost completely the myoblast shape, and reversine compared with the WT enzyme (Figure3a, and data were not able to form myotubes (Figure 4a). not shown). Furthermore, we also investigated whether the presence of Next, we used these drug-resistant mutants to directly test Aurora B, resistant to reversine, would inhibit the whole the role of Aurora B in the process of dedifferentiation induced dedifferentiation–redifferentiation process induced by rever- by reversine. We transiently transfected C2C12 cells at 30% sine. After 72 h of reversine treatment, cells were transferred of confluence with plasmids encoding either for WT human into the appropriate differentiation medium and cultured for 1 Aurora B or the Y156H and G160V mutants. After 48 h, week. Remarkably, in the presence of both mutants,

Cell Death and Differentiation Aurora B inhibition induces dedifferentiation G Amabile et al 325

control reversine

reversine + Aurora G160V reversine + Aurora WT

ALP activity Adipo Red assay 3000 2500 2500 2000 2000 1500 1500 ∗ RFU RFU 1000 1000 ∗∗ ∗∗ ∗∗ 500 ∗∗ 500

0 0 Reversine - + + + + Reversine - + + + + (50 nM) (50 nM) Aurora B Aurora B Aurora B Aurora B Aurora B Aurora B WT Y156H G160V WT Y156H G160V

C2C12 cells C2C12 cells Figure 4 Drug-resistant Aurora B kinase inhibits the dedifferentiation process showing positive evidence of differentiation. C2C12 cells were transfected with Aurora B wild type (WT) and Aurora B G160V. After 48 h, cells were treated with reversine 50 nM for further 72 h and then cultured in growth medium for additional 8 days. (a) Myotubes formation was observed for control cells, as well for the Aurora B G160V tranfected cells. C2C12 cells treated with reversine (not transfected) and cells transfected with Aurora B WT did not show any evidence of terminal differentiation. (b) C2C12 cells were transfected with Aurora B WT, Y156H and G160V variants as indicated. After the treatment (72 h), the cells were cultured in osteogenic-differentiating (ODM) and adipogenic-differentiating media (ADM) for 1 week. ALP activity and lipid droplets content were measured to evaluate the presence of osteoblasts and adipocytes, respectively using a fluorescent substrate system. Data are expressed as relative fluorescence units (RFUs; see Materials and Methods also). Values of RFUs were statistically different with (*Po0.05) and (**Po0.01)

reprograming of C2C12 myoblasts induced by reversine was segregation failure, spindle checkpoint override and drastically reduced (Figure 4b). The simplest interpretation of impairment of cytokinesis. Taken together, these effects these results is that Aurora B kinase activity is essential for the result in .24 In principle, it is conceivable that it is action of reversine. We observed that cells transfected with WT the polyploidy itself, rather than the inhibition of Aurora B per Aurora B displayed only a partial abrogation of the reversine se, which is responsible for the dedifferentiation process. effect, presumably because of a gene dosage effect. However, we feel that this is unlikely: other agents, which cause polyploidy, including the MEK1 inhibitor U0126 and the Reversine induces polyploidy and spindle checkpoint NMMII inhibitor blebbistatin do not cause reprogramming override at high concentration. The results presented so (data not shown). Moreover, reversine induces reprograming far show that Aurora B kinase is a key player in the nuclear at concentrations as low as 20–50 nM, whereas significant reprograming induced by reversine. Aurora B is involved in polyploidy is only observed at higher concentration of multiple mitotic functions, and therefore inhibiting its kinase reversine (Figure 5a). Similar considerations are true also activity causes a variety of phenotype including chromosome for the effect on the spindle check point: SP600125, a potent

Cell Death and Differentiation Aurora B inhibition induces dedifferentiation G Amabile et al 326

Control Reversine 50 nM Reversine 100 nM Reversine 1 M Reversine 5 M 2N 2N 2N 2N 4N

2N 4N 4N 8N

4N 4N

Control Nocodazol

60x 60x

30

25

20

20x 20x 15

Nocodazol + reversine 50 nM Nocodazol + reversine 1M 10 Cells in Mitosis (% of cells plated) 5

0 Nocodazol -+++++ Reversine - - 50 nM 100 nM 500 nM 1 M

20x 20x

Figure 5 Reversine causes polyploidy and spindle checkpoint overriding at high concentrations. (a) C2C12 cells were treated for 72 h with different concentration of reversine as indicated, thus cultured in ODM for additional 7 days. After this period, the cell-cycle analysis of C2C12 cell was performed using propidium Iodide with FACS Canto II. (b–c) C2C12 cells were treated with 500 nM nocodazol in presence of increasing concentrations of reversine (as indicated) for 4 h. After this time, the cells were stained with DAPI to analyze the nucleus status and the cells blocked in mitosis were counted. Red arrows in the panel b indicate cells in mitosis

spindle checkpoint overrider does not induce reprograming, suggesting that these factors play a crucial role both in the and even reversine must be used at concentrations above induction of muscle differentiation and in the maintenance of 1 mM to cause spindle checkpoint overriding in C2C12 the differentiated muscle phenotype. Reversine treatment myoblasts (Figure 5b and c). affects the expression of MRFs; in Figure 6b–c, we show an On balance, therefore, we suspect that the role of Aurora B evident downregulation of the mRNA levels coding for MyoD, in maintaining the differentiated state of C2C12 cells is not myogenin and myogenic factor-5 (Myf5) after reversine linked to its mitotic functions. More likely, the direct involve- treatment. Interestingly, we also observed that the ment of Aurora B on reversine-induced dedifferentiation is expression of Id1, a known inhibitor of myogenesis,25 based on its role in chromatin remodeling. Consequently, we increased considerably after the treatment (Figure 6b, turned our attention to another key function of Aurora B, lower right panel). The treatment of myoblasts with the namely histone H3 (Ser10) phosphorylation (phospho-Ser10- other Aurora inhibitors also caused a relevant dowregulation H3) and a possible role in chromatin remodeling. As predicted of MyoD and Myf5, although, on the other hand, appeared for a potent Aurora B inhibitor, reversine does indeed inhibit the upregulation of the Id1 expression (Figure 6c), phospho-ser10-H3 in C2C12 cells (Figure 6a). Furthermore, suggesting a common mechanism of action for the three the inhibitory effect of reversine is significantly reduced drugs, most likely triggered by the inhibition of Aurora B following the expression of the drug-resistant Aurora B kinase. In fact, reversine treatment in the presence of Aurora mutants (Figure 6a) confirming that Aurora B is the bona fide B kinase-resistant mutants did not lead to the downregulation serine 10 kinase, at least in C2C12 cells. of MyoD expression compared with the untreated cells of with cells trasfected with Aurora B WT (Figure 6b). Reversine induces remodeling of chromatin at the level Apparently, Aurora B inhibitors and reversine can cause of muscle differentiation genes. Myogenesis is a complex not only the repression but also, with similar kinetics, the process regulated by muscle regulatory factors (MRFs) and activation of genes. To elucidate the putative common their inhibitors (Ids proteins).25 As reported by several molecular event that is at the core of both activation and groups,26–28 ectopic expression of MRFs, such as MyoD or repression of genes, we analyzed by chromatin immuno- myogenin, converts nonmuscle cells into muscle cells, precipitation (ChIP) histone modifications at the level of the

Cell Death and Differentiation Aurora B inhibition induces dedifferentiation G Amabile et al 327

MyoD 1.2 1.0

Control Control Aurora B Y156HAurora B G160V 0.8 Reversine (50 nM) - + ++ 0.6 Anti-myc-Tag 0.4 Fold change m/h-Aurora B 0.2

ser10P-H3 0.0 Reversine (50 nM) - +++ Actin

Control Control

Aurora B WT Aurora B G160V

c MyoD Myogenic factor 5 (Myf5) 1.2 1.2 1 1 0.8 0.8 0.6 0.6 0.4 0.4

Fold change 0.2 0.2 0 0 Control 24 h 48 h 72 h Control 24 h 48 h 72 h

Myogenin Id1 1.2 4 1 3 0.8 0.6 2 0.4

Fold change 1 0.2 0 0 Control 24 h 48 h 72 h Control 24 h 48 h 72 h Reversine treatment (50 nM)

d MyoD Myf5 Id1 1.2 1.2 12 1.0 1.0 10

0.8 0.8 8 0.6 0.6 6 0.4 0.4 4 Fold change 0.2 0.2 2 0.0 0.0 0

Control VX-680 Control VX-680 Control VX-680 Reversine Reversine Reversine Hesperadin Hesperadin Hesperadin 72-h treatment (500 nM) Figure 6 Reversine inhibits histone H3 phosphorylation on Ser10 and induces the downregulation of MRFs expression . (a) C2C12 trasfected with Aurora B mutants were treated with 50 nM of reversine for 48 h. After the treatment, the Ser10-H3 phosphorylation status was analyzed by western blot. Anti-Myc and anti-Aurora were used as transfection control, whereas actin was used as loaded control. (b) MyoD mRNA levels were evaluated in untreated cells, cells treated with reversine and in cells previously transfected with Aurora B G160V, Aurora B wild type (WT), and then treated with reversine for 72 h. (c) C2C12 cells were treated with 50 nM of reversine for 24, 48 and 72 h. MRFs and Id1 expressions were analyzed using the real-time PCR with Taqman (MyoD) and SYBR green technologies (Myogenin, Myf5 and Id1). MyoD mRNA levels were normalized to 18S mRNA levels. Myf5, myogenin and Id1 mRNA levels were normalized using Gapdh. The untreated C2C12 cells were used as standard to calculate the fold enrichment. (d) C2C12 cells were treated with 500 nM of reversine, VX-680 and hesperadine for 72 h. MRFs and Id1 expressions were analyzed using the real-time PCR with Taqman (MyoD) and SYBR green technologies (Myogenin, Myf5 and Id1)

MyoD, Myogenin and Id1 promoters. In essence, at the level reversine dependent change, neither in the overall H3 of the repressed genes (MyoD and Myogenin), we observed acetylation, nor methylation (Lys4 and Lys9 were analyzed; the expected inhibition of phospho-Ser10 but no other Figure 7). Interestingly, but not surprisingly, the dual

Cell Death and Differentiation Aurora B inhibition induces dedifferentiation G Amabile et al 328

MyoD promoter H3 acetylated p-Ser10-H3 meLys9-H3 meLys4-H3 1 2.0 1 0.4 0.8 0.8 1.6 0.3 1.2 0.6 0.6 0.2 0.8 0.4 0.4 0.4 0.2 ∗∗ 0.2 0.1 Fold enrichment Fold 0 0 0 0 Control Reversine Control Reversine Control Reversine Control Reversine

Myogenin promoter H3 acetylated p-Ser10-H3 meLys9-H3 meLys4-H3 0.6 2.5 1.5 0.8 0.5 2 0.6 1 0.4 1.5 0.3 0.4 1 0.2 0.5 0.2 0.1 0.5 ∗∗ Fold enrichment Fold 0 0 0 0 Control Reversine Control Reversine Control Reversine Control Reversine

Id1 promoter (TATA box) H3 acetylated p-Ser10-H3 meLys9-H3 meLys4-H3

2.5 ∗∗ 4 1 1 2 3 0.8 0.8 1.5 0.6 0.6 2 1 0.4 0.4 ∗ 0.5 1 0.2 0.2

Fold enrichment Fold ∗∗ 0 0 0 0 Control Reversine Control Reversine Control Reversine Control Reversine Figure 7 Aurora B is involved in affecting chromatin status. Chromatin immunoprecipitation analysis was performed on C2C12 cells treated with reversine 50 nM. Polyclonal antibodies against H3 acetylated at lysine 9 and 14, against phospho-Ser10-H3, against dymethil Lys 9-H3 and against dymethil Lys 4-H3 were used to precipitate the protein–chromatin complex. The DNA was purified, and specific regions of mouse MyoD, Myogenin and Id1 promoters were analyzed using the real-time PCR (see Materials and Methods). Rabbit IgG was used as control of the assay specificity (purple barr). Asterisks indicate the values of fold enrichment statistically different with (*Po0.05) and (**Po0.01)

modification of acetyl Lys14 and phospho-Ser10 that repre- (increase in acetylation, decrease of Lys9 methylation). It is sent a marker of gene induction29 was drastically reduced in likely that Aurora B associates with different protein com- the genes repressed by reversine (Figure 8). This result is in plexes to mark active and repressed genes. In the future, it will agreement with the findings of Mal et al.,30 who reported that be important to elucidate the molecular bases for these gene- the presence of phospho-Ser10 at the level of myogenin specific effects. promoter is a marker of active chromatin. Therefore, it is likely that the reversine causes a decrease in the expression of the Materials and Methods myogenin and MyoD genes by inhibiting the phospho-Ser10- Cell culture, treatment and transfection. C2C12 myoblasts (ATCC H3. In contrast to the repressed genes, the pattern of histone Manassas, VA) were cultured in growth medium consisting of DMEM supplemented modification at the promoter level of the induced gene Id1 was with 2 mM glutamine, 100 IU/ml penicillin, 100 mg/ml streptomycin (Invitrogen Corp., drastically changed following the treatment with reversine. We Carlsbad, CA) and 10% (v/v) FBS (HyClone, South Logan, UT). C2C12 cells were not only observed the expected inhibition of phospho-Ser10 treated with reversine, VX-680, hesperadin, MLN-8054 at a concentration range from 20 nM to 5 mM, whereas control cells were incubated with 0.01% DMSO. but also a significant increase of overall H3 acetylation as well Treatment was maintained for 48 and/or 72 h without the growth medium changes. as a decrease of the Lys9 methylation. C2C12 cells were transiently transfected with Lipofectamine 2000 (Invitrogen) as In summary, it appears that the common feature after suggested by the manufacturer. The cells were screened for the presence of ectopic treatment with reversine is the drastic reduction of phospho- gene expression after 48 h by western blot.

Ser10-H3 on all the promoters analyzed (Figure 7), as 45–344 835–903 expected for a potent Aurora B kinase inhibitor. Surprisingly, Kinase assays. Recombinant human Aurora B and human INCENP were expressed and purified as described previously.23 Kinase assays were carried out inhibition of phospho-Ser10-H3 leads to opposite effects in Id1 32 in 30 ml reaction mix containing 50 mMATP,1mMDTT,1mMNa3VO4,5mCi [g- P]- gene (induction) and in MyoD and myogenin (repression). ATP, 5 mg of histone H3 as substrate, 1 ml DMSO or drugs dissolved in DMSO, and 17 Recently, Sabbattini et al. reported the results that are in line 5 nM of WT or G160V mutant Aurora B/INCENP. Reaction mixes were incubated for with our observations. These authors described a new role of 1 h at 301C, quenched with SDS-loading buffer and resolved on 14% SDS- 32 Aurora B kinase in the repression of genes in postmitotic polyacrylamide gel (SDS-PAGE). Incorporation of P was visualized by differentiated cells. Specifically, they reported that phospho- autoradiography. Ser10 could be a marker of gene repression. Here, We show RNA preparation and quantitative analysis. Total RNA was prepared that the Id1 gene is induced as a consequence of Aurora from C2C12 cells by using Qiagen’s RNeasys midi kit (Qiagen Inc., Valencia, CA). B inhibition, and the chromatin at the level of its promoter is MyoD expression was assessed after the treatments as indicated using TaqMant drastically remodeled with markers typical of active genes chemistry with the ABI 7700 Prism real-time PCR (RT-PCR) instrument (Applied

Cell Death and Differentiation Aurora B inhibition induces dedifferentiation G Amabile et al 329

MyoD promoter Myogenin promoter H3 acetylated H3 acetylated

1.20 0.40

0.30 0.80 0.20 0.40 0.10 Fold enrichment 0.0 0.0 Untreated Rev 50 Rev 500 Untreated Rev 50 Rev 500 cells cells 48-hr treatment 48-hr treatment

MyoD promoter Myogenin promoter p-(Ser10) acetyl-(Lys14)-H3 p-(Ser10) acetyl-(Lys14)-H3

0.08 0.04

0.06 0.03

0.04 0.02 ∗ 0.01 0.02 ∗∗ ∗∗

Fold enrichment ∗∗ 0.0 0.0 Untreated Rev 50 Rev 500 Untreated Rev 50 Rev 500 cells cells 48-hr treatment 48-hr treatment Figure 8 Reversine decreases drastically Ser10-H3 phoshorylation at levels of MRFs genes. Chromatin immunoprecipitation analsysis was performed on C2C12 cells treated with reversine 50 and 500 nM as indicated. Polyclonal antibodies against H3 acetylated at lysine 9 and 14 and against anti-phospho-Ser10-acetyl (K14)-histone H3 were used to precipitate the chromatin—protein complex. The DNA was purified and specific regions of mouse MyoD and myogenin promoter were analyzed using the real- time PCR (see Materials and Methods). Rabbit IgG was used as control of the assay specificity (purple barr). Values obtained for the fold enrichment were statistically different with (*Po0.05) and (**Po0.01)

Biosystems, Foster City, CA). The forward and reverse primer (200 nM) sequences microscope (Leica Microsystems, Wetzlar, Germany). For quantitative analysis, for mouse MyoD were 50-TTCTTCACCACACCTCTGACA-30 and 50-GCCGT after being cultured for 7 days in ADM, cells were incubated with AdipoRed reagent GAGAGTCGTCTTAACTT-3,0respectively, and the probe sequence (100 nM) was for 10 min. ALP activity was measured with a fluorescent substrate system 50-ACAGCCGGTGTGCATTCCAA-30. Data analysis was performed using 18S as (AttoPhos AP; Promega, Madison, WI). After 7 days in ODM, cells were incubated internal standard. The forward and reverse primer sequences for mouse 18S with AttoPhos Substrate (Promega, Madison, WI) for 15 min. Fluorescence was were 50-CGGCTACCACATCCAAGGAA-30 and 50-GCTGGAATTACCGCGGCT-3,0 measured as suggested by the manufacturer using Fusion Universal Microplate respectively, and the probe sequence was 50- TGCTGGCACCAGACTTGCCCTC-30. Analyzer and Envision Microplate Analyzer (Perkin Elmer). Mouse Myf5 and Myogenin were analyzed as reported previously,31 whereas Id1 32 was analyzed as reported by Gersbach et al. The expression was measured using Western blot analysis. Cells were lysed in 50 mM Tris/HCl buffer, pH 8.0, SYBR green chemistry with the ABI 7700 Prism RT-PCR instrument (Applied containing 150 mM NaCl, 1% Nonidet P-40, 2 mg/ml aprotinin, 1 mg/ml pepstatin, Biosystems). The forward and reverse primer sequences of mouse Gapdh used as 2 mg/ml leupeptin and 1 mM Na VO . Equal amounts of extracted proteins (50 mg) 0 0 0 3 4 internal standard were 5 -GGAGATTGTTGCCATCAACGACC-3 and 5 -GGTCAT- were loaded and separated on SDS-PAGE (Invitrogen), and then transferred onto a GAGCCCTTCCACAATGC-3,0 respectively. Osteopontin, collagen type 1, ap2 and 18 nitrocellulose membrane (Schleicher & Schuell Biosciences, Sandford, ME). LPL expressions were analyzed with SYBR green method as reported previously. Immunodetection was performed using the following primary antibodies: anti- Myc-Tag and anti- ser10P-H3 (Millipore, Billerica, MA), antimouse Aurora B (Becton Dickinson, San Jose, CA) and antimouse/human Aurora B (Abcam Inc., Dedifferentiation assay. C2C12 cells were cultured in the presence of Cambridge, MA). reversine, VX-680, hesperadin, MLN-8054 as described. After 72 h of treatment, compounds were removed and cells were cultured in (ADM or in ODM. Adipogenic- inducing medium contained DMEM 10% FBS, 4.5 g/l glucose, 1 mM analysis. C2C12 cells were incubated with reversine as indicated dexamethasone, 0.5 mM isobutylmethylxantine and 10 mg/ml insulin (Sigma- for 48 h, then the growth medium contained reversine was changed and the cells Aldrich, St. Louis, MO), whereas osteogenic-inducing medium contained DMEM- were cultured with a medium suitable for the development of osteoblstasts (ODM) 10% FBS, 50 mg/ml ascorbic acid-2-phosphate, 0.1 mM dexamethasone and 10 mM for 96 h. Cells were fixed in 70% ethanol over night. After double washing with PBS, b-glicerophosphate (Sigma). For hystochemical analysis, after 7 days of culture in cells were labeled with cell–cycle-staining reagent PBS/0.In all, 1% Triton X-100, ADM or ODM, cells were fixed with 4% paraformaldehyde for 15 min and washed 200 mg/ml DNAse-free RNAse, 25 mg/ml propidium iodide (Invitrogen) and twice with PBS. Cells were then incubated for 2 h with Oil Red O reagent (Sigma) to incubated at room temperature, in the dark, for 30 min. DNA content was evaluate the presence of adipocytes and with mixture of 0.1 mg/ml of naphthol analyzed using FACS Canto II (Becton Dickinson). AS-MX phosphate (Sigma), 0.5% N,N-dimethylformamide, 2 mM MgCl2 and 0.6 mg/ ml of fast blue BB salt (Sigma) in 0.1 M Tris-HCl, pH 8.5, to evaluate the presence of Immunofluorescence. In all, 5 Â 104 cells were cultured on poly-lysine osteoblasts. Cells were examined by phase-contrast microscopy with DFC320 (Sigma)-coated coverslips and incubated in the presence of nocodazol 500 nM and

Cell Death and Differentiation Aurora B inhibition induces dedifferentiation G Amabile et al 330

reversine as indicated for 4 h. Cells were fixed in 4% paraformaldehyde for 20 min at 11. Vader G, Cruijsen CW, van Harn T, Vromans MJ, Medema RH, Lens SM. The room temperature. After two washes with PBS, cells were stained with DAPI 0.5 mg/ chromosomal passenger complex controls spindle checkpoint function independent ml for 1 h. The samples were analyzed with Confocal Microscopy with LSM 510 from its role in correcting interactions. Mol Biol Cell 2007; 18: META microscope (Zeiss, Thornwood, NY). 4553–4564. 12. Musacchio A, Salmon ED. The spindle-assembly checkpoint in space and time. Nat Rev 6 Mol Cell Biol 2007; 8: 379–393. Chromatin immunoprecipitation. Cells (10 for each antibody) were used 13. Fischle W, Tseng BS, Dormann HL, Ueberheide BM, Garcia BA, Shabanowitz J et al. to cross-link chromatin using the protocol from Upstate Biotechnology (Milton Regulation of HP1-chromatin binding by histone H3 methylation andphosphorylation. Keynes, UK) with minor modifications. Briefly, cells were incubated in 1% Nature 2005; 438: 1116–1122. formaldehyde for 10 min at room temperature. Glycine (0.125 M) was added to stop 14. Terada Y. Aurora-B/AIM-1 regulates the dynamic behavior of HP1alpha at the G2-M cross-linking. Cross-linked chromatin was sonicated three times for 10 s at 80% transition. Mol Biol Cell 2006; 17: 3232–3241. amplitude (Vibra-Cell sonicator; Sonics and Materials, Meryin/Satigny, Switzerland). 15. Jenuwein T, Allis CD. Translating the histone code. Science 2001; 293: 1074–1080. Polyclonal antibodies raised against H3 acetylated at lysine 9 and 14 (aH3-Ac) and 16. Williams L, Zhao J, Morozova N, Li Y, Avivi Y, Grafi G. Chromatin reorganization against antiphospho-S10-acetyl (K14) histone H3, both from Millipore, whereas accompanying cellular dedifferentiation is associated with modifications of histone H3, redistribution of HP1, and activation of E2F-target genes. Dev Dyn 2003; 228: 113–120. antihistone H3 dymethil K4, antihistone H3 dymethil K9 and anti-phospho-Ser10-H3 17. Sabbattini P, Canzonetta C, Sjoberg M, Nikic S, Georgiou A, Kemball-Cook G et al. A novel were purchased from Abcam. DNA was purified by phenol–chloroform extraction, role for the Aurora B kinase in epigenetic marking of silent chromatin in differentiated and specific regions of the indicated promoters were amplified by RT-PCR using postmitotic cells. EMBO J 2007; 26: 4657–4669. SYBR green method (Applied Biosystem). The primers used for mouse MyoD 18. Chen S, Takanashi S, Zhang Q, Xiong W, Zhu S, Peters EC et al. Reversine increases the promoter were 50-CGCCCCCAGCCTCCCTTTCCA-30 and 50-TGTCAGAGGTG plasticity of lineage-committed mammalian cells. Proc Natl Acad Sci USA 2007; 104: TGGTGAAGAAA-30; mouse myogenin promoter was analyzed as reported 10482–10487. previously.30 The primers used to analyze the TATA box of mouse Id1 promoter 19. Cohen P, Goedert M. GSK3 inhibitors: development and therapeutic potential. Nat Rev were 50-CTTATAAAAGACTGGCTCCAGC-30 and 50-GGAGGCTGAGAACA Drug Discov 2004; 3: 479–487. 20. Hauf S, Cole RW, LaTerra S, Zimmer C, Schnapp G, Walter R et al. The small molecule GAAACAGAGTGTG-30 To calculate the fold enrichment, a previously published 33 hesperadin reveals a role for Aurora B in correcting kinetochore–microtubule attachment method was used. and in maintaining the spindle assembly checkpoint. J Cell Biol 2003; 161: 281–294. 21. Harrington EA, Bebbington D, Moore J, Rasmussen RK, Ajose-Adeogun AO, Nakayama T Statistical analysis. Statistical analysis was performed by the analysis of et al. VX-680, a potent and selective small-molecule inhibitor of the Aurora kinases, variance (ANOVA) test using Origin 6.1 software for Windows (Microcal Software suppresses tumor growth in vivo. Nat Med 2004; 10: 262–267. Inc., Northempton, MA). 22. Manfredi MG, Ecsedy JA, Meetze KA, Balani SK, Burenkova O, Chen W et al. Antitumor activity of MLN8054, an orally active small-molecule inhibitor of . Proc Natl Acad Sci USA 2007; 104: 4106–4111. Acknowledgements. We are very grateful to Dr. Fabio Formiggini and to 23. Girdler F, Sessa S, Patercoli S, Villa F, Musacchio A, Taylor S. Molecular basis of drug-resistance in Aurora kinases. Chem Biol 2008; 15: 552–562. Dynamic Imaging Microscopy facility of CEINGE Biotecnologie Avanzate for the 24. Ditchfield C, Johnson VL, Tighe A, Ellston R, Haworth C, Johnson T. Aurora B couples help with confocal microscopy. SST is a Research UK senior fellow. chromosome alignment with by targeting BubR1, Mad2, and Cenp-E to . J Cell Biol 2003; 161: 267–280. 25. Jen Y, Weintraub H, Benezra R. Overexpression of Id protein inhibits the muscle 1. Hochedlinger K, Jaenisch R. Nuclear reprogramming and pluripotency. Nature 2006; 441: differentiation program: in vivo association of Id with E2A proteins. Genes Dev 1992; 6: 1061–1067. 1466–1479. 2. Cowan CA, Atienza J, Melton DA, Eggan K. Nuclear reprogramming of somatic cells after 26. Braun T, Buschhausen-Denker G, Bober E, Tannich E, Arnold HH. A novel human muscle fusion with human embryonic stem cells. Science 2005; 309: 1369–1373. factor related to but distinct from MyoD1 induces myogenic conversion in 10T1/2 3. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and fibroblasts. EMBO J 1989; 8: 701–709. adult fibroblast cultures by defined factors. Cell 2006; 126: 663–676. 27. Davis RL, Weintraub H, Lassar AB. Expression of a single transfected cDNA converts 4. Wernig M, Meissner A, Foreman R, Brambrink T, Ku M, Hochedlinger K et al. In vitro fibroblasts to myoblasts. Cell 1987; 51: 987–1000. reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 2007; 28. Tapscott SJ, Davis RL, Thayer MJ, Cheng PF, Weintraub H, Lassar AB. MyoD1: a nuclear 448: 318–324. phosphoprotein requiring a Myc homology region to convert fibroblasts to myoblasts. 5. Maherali N, Sridharan R, Xie W, Utikal J, Eminli S, Arnold K et al. Directly reprogrammed Science 1988; 242: 405–411. fibroblasts show global epigenetic remodeling and widespread tissue contribution. Cell 29. Clayton AL, Rose S, Barratt MJ, Mahadevan LC. Phosphoacetylation of histone H3 on Stem Cell 2007; 1: 55–70. c-fos- and c-jun-associated upon gene activation. EMBO J 2000; 19: 6. Odelberg SJ, Kollhoff A, Keating MT. Dedifferentiation of mammalian myotubes induced by 3714–3726. msx1. Cell 2000; 103: 1099–1109. 30. Mal A, Harter ML. MyoD is functionally linked to the silencing of a muscle-specific 7. Chen S, Zhang Q, Wu X, Schultz PG. Ding dedifferentiation of lineage-committed cells by a regulatory gene prior to skeletal myogenesis. Proc Natl Acad Sci USA 2003; 100: small molecule. J Am Chem Soc 2004; 126: 410–411. 1735–1739. 8. Anastasia L, Sampaolesi M, Papini N, Oleari D, Lamorte G, Tringali C et al. Reversine- 31. Calhabeu F, Lafont J, Le Dreau G, Laurent M, Kazazian C, Schaeffer L et al. NOV/CCN3 treated fibroblasts acquire myogenic competence in vitro and in regenerating skeletal impairs muscle cell commitment and differentiation. Exp Cell Res 2006; 312: 1876–1889. muscle. Cell Death Differ 2006; 13: 2042–2051. 32. Gersbach CA, Byers BA, Pavlath GK, Garcı´a AJ. Runx2/Cbfa1 stimulates 9. D’Alise AM, Amabile G, Iovino M, Di Giorgio FP, Bartiromo M, Sessa F et al. Reversine, a transdifferentiation of primary skeletal myoblasts into a mineralizing osteoblastic novel Aurora kinases inhibitor, inhibits colony formation of human acute myeloid phenotype. Exp Cell Res 2004; 300: 406–417. cells. Mol Cancer Ther 2008; 7: 1140–1149. 33. Litt MD, Simpson M, Recillas-Targa F, Prioleau MN, Felsenfeld G. Transitions in histone 10. Barr AR, Gergely F. Aurora-A: the maker and breaker of spindle poles. J Cell Sci 2007; 120: acetylation reveal boundaries of three separately regulated neighboring loci. EMBO J 2987–2996. 2001; 20: 2224–2235.

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Cell Death and Differentiation